Alteration of a figure to help facilitate teaching and learning about Golden Gate cloning

I make extensive use of key publications to help people understand the tools that I use for building plasmids via Type IIS restriction endonuclease mediated assembly (Golden Gate cloning). For some time I have desired to make modifications to Figure 2 from Engler et al.,  ACS Synthetic Biology (2014) v3 839-843. While the original cartoon is a good starting point for explaining the ins and outs of Golden Gate, on more than one occasion I have had the person I am speaking with become completely confused about the 3′ end of the illustrated molecules. I finally have made modifications to the image, and I hope that these will help clarify that the convention used in the Golden Gate world, whereby the “motif” or “fusion” between two pieces of DNA refers ONLY to the top strand, while the actual sequence of the 5′ overhang that is produced by BsaI digestion at the 3′ end of the molecules is actually the reverse complement of that which is shown. Additionally, I tried to impart some sense of directionality to the Type IIS enzymes by inverting the labels for those recognition motifs which reside in the bottom strand of the DNA. Finally, since color printers tend to produce a bit darker result than what one views on a computer screen, I opted to exchange the black text for white in selected colored bars used for the Level 0 components. Hopefully this will be useful for your comprehension of Golden Gate cloning.

Retouch Fig 2 ACS Synth Bio 3 (11) pp 839–843

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